Journal: Immunity

Article Title: Intestinal epithelial Wnt signaling mediates acetylcholine-triggered host defense against infection

doi: 10.1016/j.immuni.2018.04.017

Figure Lengend Snippet: Infection and acetylcholine induce Wnt ligand in the intestinal epithelium (A) qRT-PCR of Wnt pathway genes in wild type animals after 16 h S. aureus infection. Results are normalized to wild type animals fed on E. coli OP50. Data are mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). (B) Representative epifluorescence micrographs of animals expressing Venus-tagged CWN-2 protein from the cwn-2 promoter ( cwn-2p::cwn-2::venus ) after 24 h of feeding on E. coli or infection with S. aureus. (C) Quantitative analysis of (B). Results are normalized to E. coli controls. Data are mean ± SEM (two independent biological replicates, n ≥ 50 per condition). (D) Representative epifluorescence micrographs of animals expressing GFP from the mig-1 promoter ( mig-1p::gfp ) after 24 h of feeding on E. coli or infection with S. aureus. (E) Quantitative analysis of (D). Results are normalized to E. coli controls. Data are mean ± SEM (two independent biological replicates, n ≥ 50 per condition). (F) qRT-PCR of cwn-2 in wild type animals after 16 h treatment with 5 mM arecoline. Data are mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). (G) Same as in (F), but after 16 h treatment with 1 mM oxotremorine. (H) Representative epifluorescence micrographs of cwn-2p::cwn-2::venus animals after 24 h treatment with 5 mM arecoline. (I) Quantitative analysis of (H). Results are normalized to vehicle controls. Data are mean ± SEM (two independent biological replicates, n ≥ 50 per condition). (J) qRT-PCR of cwn-2 in wild type animals and unc-17 mutants infected with S. aureus for 16 h. Results are normalized to E. coli wild type control. Data are mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). (K) CWN-2::Venus expression after 24 h S. aureus infection, in animals reared on E. coli carrying empty vector or RNAi against gar-2 and gar-3 , egl-30, egl-8, or pkc-1 . Data are mean ± SEM (two independent biological replicates, n ≥ 50 per condition). Results are normalized to E. coli empty vector control. (L) qRT-PCR of cwn-2 in wild type and egl-8 mutant animals after 16 h treatment with 5 mM arecoline. Results are normalized to vehicle-treated wild type. Data are mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). (M) Time course of aldicarb paralysis. Data are the ratio of paralysis frequencies of infected animals v. E. coli controls at each time point. Results are representative of 3 independent biological replicates. (N) Time course of cwn-2 and clec-60 mRNA induction. Data are cwn-2 or clec-60 expression in infected animals relative to E. coli controls. Mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). (O) Schematic summary of results. Scale bars, 0.6 mm. Scale bars with *, 0.2 mm.

Article Snippet: Arecoline hydrobromide (Sigma, 31593-250MG), 1 mM for killing assays and 5 mM for other experiments; Scopolamine (Sigma PHR1470-500MG), 1 mM for killing assays and 5 mM for other assays; Oxotremorine (sigma, O100-100MG) 1 mM, LiCl (Sigma 203637-10G) 100 mM).

Techniques: Infection, Quantitative RT-PCR, Expressing, Plasmid Preparation, Mutagenesis

Journal: Immunity

Article Title: Intestinal epithelial Wnt signaling mediates acetylcholine-triggered host defense against infection

doi: 10.1016/j.immuni.2018.04.017

Figure Lengend Snippet: Muscarinic receptors control host defense against infection (A) Epifluorescence micrographs of animals expressing GFP from the clec-60 promoter ( clec-60p::gfp ) after 8 h of feeding on E. coli, starvation, or infection with S. aureus. (B) Quantitative analysis of (A). Data are mean ± SEM (two independent biological replicates, n ≥ 50 per condition). ** p ≤ 0.01 (see STAR★Methods for description of statistical methods). ns, not significant. (C) Representative epifluorescence micrographs of clec-60p::gfp animals that were reared on E. coli carrying empty vector (top row) or gar-2 , gar-3 double RNAi (bottom row), and subsequently infected 16 h with S. aureus (left column) or treated for 16 h with 5 mM arecoline (right column). (D) Quantitative analysis of (C). Data are mean ± SEM (two independent biological replicates, n ≥ 50 per condition). (E) clec-60p::gfp expression after 16 h incubation of uninfected animals with 5 mM arecoline. Data are mean ± SEM (at least two independent biological replicates, n ≥ 50 per condition). (F) qRT-PCR of clec-60 , ilys-3, and lys-5 in wild type animals incubated with 5 mM arecoline for 8 h, normalized to vehicle treated animals. Data are mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). * p ≤ 0.05. (G) Representative epifluorescence micrographs of clec-60p::gfp animals that were treated with vehicle (top row) or 5 mM scopolamine (bottom row), during 16 h infection with S. aureus (left column) or treated with 5 mM arecoline for 16 h (right column). (H) Quantitative analysis of infection in (G). (I) Quantitative analysis of scopolamine treatment in (G). Data are mean ± SEM (at least two independent biological replicates, n ≥ 50 per condition). (J) qRT-PCR of clec-60 in wild type animals treated with 1 mM oxotremorine for 8 h. Results are normalized to vehicle-treated animals. Data are mean ± SEM (three independent biological replicates, n ≥ 3,000 per condition). (K) Survival of wild type animals, treated with vehicle or 1 mM arecoline for 24 h prior to infection. Results are representative of 2 independent biological replicates. *** p ≤ 0.001. (L) Lifespan of wild type animals treated with vehicle or 1 mM arecoline for 24 h before transfer to E. coli OP50. Results are representative of 2 independent biological replicates. ns, p > 0.05. (M) Survival of wild type animals, treated with vehicle or 1 mM scopolamine during the entire course of infection. Results are representative of 2 independent biological replicates. (N) Lifespan of wild type animals on E. coli OP50, treated with vehicle or 1 mM scopolamine. Results are representative of 2 independent biological replicates. (O) Survival of wild type and unc-17 mutant animals infected with S. aureus . Results are representative of 2 independent biological replicates. (P) Aldicarb paralysis assays of infected and uninfected animals. Results are representative of at least 3 independent biological replicates. (Q) .

Article Snippet: Arecoline hydrobromide (Sigma, 31593-250MG), 1 mM for killing assays and 5 mM for other experiments; Scopolamine (Sigma PHR1470-500MG), 1 mM for killing assays and 5 mM for other assays; Oxotremorine (sigma, O100-100MG) 1 mM, LiCl (Sigma 203637-10G) 100 mM).

Techniques: Infection, Expressing, Plasmid Preparation, Incubation, Quantitative RT-PCR, Mutagenesis

Journal: bioRxiv

Article Title: SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic β-cells

doi: 10.1101/2020.01.11.902916

Figure Lengend Snippet: SPARC enhanced muscarinic receptor agonist-stimulated insulin secretion. (A) Mouse islets were isolated and culture in RPMI1640 containing 11 mM glucose overnight. The islets were infected with Ad-GFP or Ad-SPARC for 24 hrs. The islets were stimulated with 16.7 mM glucose, or 16.7 mM glucose and 100 µM Oxo-M in Krebs Ringer Bicarbonate buffer for 1 hr. The supernatants were collected and insulin was determined with insulin ELISA kit. The results were derived from three individual experiments. * denotes P

Article Snippet: Oxotremorine M (Oxo-M) and LY294002 were purchased from Sigma-Aldrich.

Techniques: Isolation, Infection, Enzyme-linked Immunosorbent Assay, Derivative Assay

Journal: bioRxiv

Article Title: SPARC promotes insulin secretion through down-regulation of RGS4 protein in pancreatic β-cells

doi: 10.1101/2020.01.11.902916

Figure Lengend Snippet: RGS4 inhibitor CGG-4986 restored insulin secretion in islets of sparc -/-mice. (A) Mouse islets were isolated from WT or sparc -/- mice. The isolated islets were cultured for 16 hrs in RPMI1640 with 11 mM Glucose and 10% FBS. The WT or sparc -/- islets were incubated for 1 hr in 2.8 mM glucose, then the medium was changed to 16.7 mM glucose, 16.7 mM glucose with Oxo-M, 100 µM in Krebs Ringer Bicarbonate buffer for 1 hour. The supernatants were collected for insulin assay. Shown were the levels of insulin secretion normalized to total protein concentrations. * denotes P

Article Snippet: Oxotremorine M (Oxo-M) and LY294002 were purchased from Sigma-Aldrich.

Techniques: Mouse Assay, Isolation, Cell Culture, Incubation